Heat‐killed Lactobacillus murinus confers neuroprotection against dopamine neuronal loss by targeting NLRP3 inflammasome

Abstract The intestinal flora has become very active in studies related to Parkinson's disease (PD) in recent years. The microbe‐gut‐brain axis is closely related to the maintenance of brain homeostasis as well as PD pathogenesis. Alterations in gut bacteria can contribute to neuroinflammation and dopamine (DA) neurodegeneration. Lactobacillus murinus, a gram‐positive bacterium, is a commensal gut bacteria present in the mammalian gut and considered as a potential probiotic due to its beneficial effects, including anti‐inflammatory and antibacterial actions. In this study, the effects of live L. murinus and heat‐killed L. murinus on DA neuronal damage in rats and the underlying mechanisms were investigated. Data showed that heat‐killed L. murinus ameliorated 6‐hydroxydopamine‐induced motor dysfunctions and loss of substantia nigra DA neurons, while no protection was shown in live L. murinus treatment. At the same time, heat‐killed L. murinus reduced the activation of NLRP3 inflammasome in microglia and the secretion of pro‐inflammatory factors, thus inhibiting the development of neuroinflammation. Furthermore, heat‐killed L. murinus failed to display its original neuroprotective properties in NLRP3 inflammasome knockout mice. Together, heat‐killed L. murinus conferred neuroprotection against DA neuronal loss via the inhibition of microglial NLRP3 inflammasome activation. These findings provide a promising potential for future applications of L. murinus, and also beneficial strategy for PD treatment.

60 years old. 1,2 Degeneration and loss of dopamine (DA) neurons in substantia nigra was the main pathological feature of PD patients.
With the progression of the disease, motor symptoms (tremor, stiffness, bradykinesia) and non-motor symptoms (cognitive decline, constipation, and sleep disturbance) superimpose. 3,4 Although the exact pathogenesis of PD remains to be elucidated, studies on a large number of PD patients, animal models and cell experiments have shown that neuroinflammation plays an important role in the occurrence and development of PD. [5][6][7] Microglia are important innate immune cells in the central nervous system (CNS). When inflammation or other pathogenic factors attack the nervous system of the brain, microglia respond rapidly, and the activated microglia change their morphology and release various inflammatory mediators, such as IL-1β, IL-18, and TNF-α. Studies have shown that these cytokines are involved in mediating neuroinflammation and acute or chronic neurodegeneration in CNS pathology. IL-1β, normally expressed at low levels, activates microglia when secreted in large quantities. 8,9 Moreover, IL-18 can induce interferon-γ through natural killer cells, guide autoreactive T cells, and promote autoimmune neurodegeneration in CNS. 10 TNF-α in physiological state participates in homeostasis regulation and synaptic plasticity. However, massively TNF-α production induces neuronal damage through excessive release of glutamate and inhibition of astrocytic glutamate reuptake, resulting in neuronal excitotoxicity and death. 9 The secretion of these cytokines is closely related to the NOD-like receptor protein 3 (NLR family pyrin domain containing 3, NLRP3) inflammasome in microglia. Current evidence indicates that the NLRP3 inflammasome complex is upregulated in microglia of the substantia nigra in PD patients and PD animal models. 11,12 In addition, NLRP3 sensor, apoptosis-associated card-containing speck-like protein (ASC) and caspase-1 form the inflammatory complex, 13,14 resulting in caspase-1-dependent activation and secretion of the pro-inflammatory cytokines, IL-1β, and IL-18. 15 These pro-inflammatory factors further stimulate the activation of microglia, leading to persistent and slow neuroinflammation in the brain. Inhibition of the NLRP3 inflammasome signaling activation could alleviate the progression of PD. 16,17 Therefore, the NLRP3 inflammasome in microglia might be an effective target for PD therapy.
Research on the intestinal flora in relation to neurodegenerative diseases, such as PD, has become very active in recent years. The microbe-gut-brain axis is closely related to the maintenance of brain homeostasis as well as PD pathogenesis. 18,19 Alterations in gut bacteria can contribute to neuroinflammation and DA neurodegeneration by promoting enteric and peripheral neurogenic/inflammatory responses. 20 In particular, NLRP3 inflammasome actively participates in or shapes peripheral and central immune/inflammatory responses in CNS diseases. NLRP3 inflammasome activation affected gut microbial composition, and gut microbial dysbiosis could also lead to overactivation of the NLRP3 inflammasome pathway. [21][22][23] Clinical studies have shown that the gut microbiome was altered in PD patients, in addition to the activation of the NLRP3 inflammasome in the gut, periphery, and brain, which may suggest that microbes, NLRP3 inflammasome and PD interact with each other. 24 Today, a growing body of studies confirms the beneficial effects of probiotics and heat-killed microorganisms on the host. Probiotics or inactivated microorganisms can play an immunomodulatory role in modulating NLRP3 inflammasome activity. 25,26 As COVID-19 swept the world in the past 2 years, probiotics have been discussed as a potential treatment for 28 In addition, inactivated microorganisms have been considered as candidate immunomodulators for COVID-19. 29 Thus, probiotics or inactivated microbial preparations have been proposed as therapeutic or prophylactic preparations. Collectively, the development of probiotics or inactivated microorganisms for the treatment of PD might have promising applications.
In this study, high-throughput sequencing technology was first  30,31 This suggests that L. m could have potential neuroprotective properties. However, the underlying mechanism is unclear. In the present study, 6-OHDA-induced rat DA neuronal loss was applied to explore the neuroprotective effects of live L. m and heat-killed L. m (H-k L. m) and the possible mechanisms. These findings would provide a promising potential for future applications of L. m and also beneficial strategy for PD treatment.

| Animals and treatment
Male Sprague-Dawley rats (180-220 g) were purchased from Beijing HFK Bioscience Co., Ltd and NLRP3 knockout mice were from Jiangsu Aniphe Biolaboratory Inc. Animals were raised in an environment where the temperature was maintained at 19-24 C and the humidity was kept in 40-70% with free access to food and water. Mice were genetically identified before being used in experiments. Rats were used for adaptive feeding for 7 days before experiment.

| 16S rRNA sequencing
After the modeling period, the rat feces were collected in sterile centrifuge tubes and stored at À80 C for analysis. Sample DNA was extracted by cetyltrimethyl ammonium bromide method. Appropriate amount of sample DNA was put into the centrifuge tube and diluted to 1 ng/μl with sterile water. Using the diluted genomic DNA as the template, specific primers, such as Barcode, Phusion high-fidelity PCR Master Mixwith New England Biolabs GC Buffer were used. Based on the selection of sequencing regions, PCR was performed with high fidelity enzyme. PCR products were detected by 2% agarose gel electrophoresis. The library was constructed using TruSeqDNA PCR-free sample preparation kit library construction kit, and the constructed library was quantified by qubits and Q-PCR. After the library was qualified, NovaSeq 6000 was used for sequencing on the machine.

| Metagenomics
DNA concentrations were determined using the Qubit dsDNA assay kit after DNA extraction from fecal samples, with OD values between 1.8 and 2.0 and DNA content above 1 μg to construct libraries. DNA was randomly interrupted into fragments of approximately 350 bp in length using a Covaris ultrasonic disruption instrument, and PCR products were purified by end repair, A-tail addition, and sequencing adapters (AMPure XP system). Initial quantitation was performed using Qubit 2.0, diluting the library to 2 ng/μl, followed by detection of inserts into the library using Agilent 2100, and accurate quantitation of the effective concentration of the library (effective concentration of the library >3 nM) was performed using Q-PCR method after inserts were as expected. The index-encoded samples were clustered on a cBot cluster generation system. After cluster generation, Illumina PE150 sequencing was performed and paired-end reads were generated.

| Rotarod test
Rotarod test is a common test applied to assess animal behavior dysfunctions. In detail, animals were placed on the rotating shaft rod of the fatigue meter, and the corresponding acceleration parameters were selected for the experiment (4.0-40 rpm/0-30 rpm) until animals fell off the rotating rod. The exercise ability of animals was measured according to the setting program of fatigue instrument, and the time animals stayed on rod was recorded. Each animal was tested for three times with an interval of more than 30 min. The mean value was used for statistical analysis.

| Forepaw adjusting steps test
Forepaw adjusting steps test was conducted 1 d after last L. m treatment. In detail, the posterior two legs and one forepaw of rat were fixed so that the rat body weight was supported by only one forelimb (the posterior two legs were suspended by lifting the tail of mouse and the body was supported by two forepaws). Rats were moved 90 cm forward at a constant speed within 10 s (mice were moved 90 cm backward at a constant speed within 4 s). The number of adjusted steps of the rat/mouse forelimb was recorded, and the number of adjusted steps of the other forelimb was measured by the rat simultaneous method. The test was repeated three times and results were represented by (number of moving steps on the injured side/ number of moving steps on the injured side) Â 100%.

| Western blotting
Animals were sacrificed after deep anesthesia and brains were quickly removed and midbrain tissues were isolated on ice and cryopreserved at À80 C. The tissue was homogenized by adding lysis buffer (containing protease inhibitors), and the supernatant collected by centrifugation after lysis on ice was the proprotein solution. The stock solution was diluted 50-fold and quantified using a BCA protein quantification kit. After adding protein stock solution, PBS and loading buffer to centrifuge tubes, the samples used for immunoblotting were denatured at 100 C. Equal amount of sample was loaded on sodium dodecyl sulfate polyacrylamide electrophoresis gel (10 or 12.5%), electrophoresis parameters were first set as 70 V, and then pressurized to 110 V after 50 min. According to the target molecular weight, the protein was transferred to PVDF membrane and sealed with 5% skim milk for 2 h. The following primary antibodies were incubated at 4 C  antibodies. After PBS rinsing, the slides were mounted with glycerol.
All the results were analyzed and processed with ImageJ software after photography.

| Statistical analysis
Data were showed as mean ± standard error of the mean (SEM) using GraphPad Prism software. Statistical significance between two groups was analyzed by unpaired t-test. Significance among multiple groups was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test. Values of p < 0.05 were considered statistically significant. In addition, the correlation between the degree of TH injury and the level of IBA-1 activation as well as the level of IBA-1 and NLRP3 activation was tested by Pearson correlation analysis.

| Changes of gut bacteria in PD rat models
Three common rat PD models were prepared by midbrain injection of LPS or 6-OHDA as well as subcutaneous injection of ROT in the back of the neck. 16S rRNA sequencing technology was used to detect the changes of gut bacteria in these 3 models. 16S rRNA sequencing technology was based on Illumina Nova sequencing platform sequencing to construct PCR-free libraries, followed by paired-end sequencing, clustering sequences into Operational Taxonomic Units (OTUs) with 97% consistency, and then species annotation of OTUs sequences with the Silva132 database. As a result, Figure 1a presented the species relative abundances of gut bacteria in 3 PD rat models, which were made based on the results of species annotation, showing the top 10 abundances of bacteria at phylum level.
Firmicutes and Bacteroidetes were seen to be the most predominant phyla among rat gut bacteria, accounting for the predominant relative abundance in all samples. Although the sequencing results indicated that the gut bacteria of three PD models exhibited a decreasing trend of Firmicutes and an increasing trend of Bacteroidetes, these changes were most obvious in 6-OHDA model than LPS or ROT models.
Next, to explore the characteristic changes of gut bacteria in PD rat models, a comparative analysis of microbial community composition was performed based on species annotation results and abundance information of OTUs from all samples. As shown in Figure 1b, nonmetric multidimensional scaling (NMDS) statistical analysis was performed for each of these three PD models. NMDS was used to investigate the differences of samples and groups. The results of NMDS analysis based on OTUs levels indicated that 6-OHDA had gradually formed a microbial community different from the control group, while LPS and ROT did not form a significantly independent community. Furthermore, Figure 1c was the result of the unweighted pair-group method with arithmetic mean (UPGMA) cluster tree analysis. According to the OTU analysis results of each sample, we used different algorithms to measure the dissimilarity coefficient between the two samples. The ordinate value corresponding to the branch node was the distance between the two samples/ clusters. If the value was lower, the diversity difference between the two samples was smaller. Therefore, the similarity relationship of gut microbiota composition between two groups could be judged by UPGMA clustering tree. Consistent with the results of NMDS analysis, the UPGMA clustering results demonstrated that the control group corresponding to 6-OHDA group showed clusters respectively, indicating that the species composition and structure of the groups were similar. However, there was no significant difference of clustering between ROT or LPS and control groups.
To further determine whether the changes in the diversity of intestinal bacteria were statistically significant, α-diversity statistical analysis was performed. Chao 1 index and ace index were used to assess species richness (OTU number). Shannon index and simpson index were used to comprehensively evaluate species richness and evenness. The results showed that chao 1 index and ace index were significantly increased in the 6-OHDA group compared with the control group (Figure 2a,b). In addition, shannon index increased significantly in the 6-OHDA group compared with the control group, while simpson index showed no significant difference (Figure 2c,d). In addition, there were no statistically significant changes in the four diversity indexes in LPS and ROT groups. These results suggest that 6-OHDA treatment can significantly change the abundance and diversity of intestinal flora in rats.

| L. m changed apparently in the 6-OHDA model
The above results showed that compared with LPS and ROT models, gut microbiota in 6-OHDA model changed obviously. Next, metagenomic sequencing was performed on 6-OHDA model. As shown in Figure 3a, changes in the relative abundance of the top 10 species at the species level in 6-OHDA group compared to control group were exhibited. Furtherly, UPGMA clustering analysis showed good clustering ( Figure 3b). Also, results of NMDS analysis matched those of 16S rRNA analysis. The 6-OHDA group was different from the control group, and these two groups formed independent microbial communities (Figure 3c). Using LDA Effect Size analysis, it could be seen from the LDA value distribution histogram that a variety of bacteria was present with significant differences between these two groups. Collectively, L. m, the bacterium with the most significant changes, was selected for potential as a probiotic in the following studies ( Figure 3d).   At the same time, H-k L. m also inhibited the protein expressions of F I G U R E 4 Effects of L. m on 6-hydroxydopamine (6-OHDA)-induced dopamine (DA) neurotoxicity. Rat behavioral changes were assessed by the rotarod test (a) and the stepping test (b) 1 day after the last administration of live L. m or H-k L. m. Brain tissues were collected after rats were sacrificed. DA neurons were labeled by immunohistochemical staining with an anti-tyrosine hydroxylase (TH) antibody (c). The "ellipses" indicated areas of substantia nigra. The loss of DA neurons in the substantia nigra was analyzed by counting the number of TH-positive neurons. Scale = 100 μm. TH protein expression was determined by western blotting (d). Data were expressed as mean ± SEM from five to eight rats. *p < 0.05 compared with control group; # p < 0.05 compared with 6-OHDA group.
F I G U R E 5 H-k L. m exerted neuroprotection against 6-hydroxydopamine (6-OHDA)-induced dopamine (DA) neurotoxicity. Behavioral changes of rats were assessed by the rotarod test (a) and the stepping test (b) 1 day after the last administration of H-k L. m. DA neurons were labeled by immunohistochemical staining (c). The "ellipses" indicated the areas of substantia nigra. The loss of DA neurons in the substantia nigra was analyzed by counting the number of tyrosine hydroxylase (TH)-positive neurons. Scale = 100 μm. TH protein expression was determined by western blotting (d). Data were presented as mean ± SEM from five to nine rats. *p < 0.05 compared with control group; # p < 0.05 compared with 6-OHDA group.

| H-k L. m inhibited NLRP3 inflammasome activation in microglia
It was well known that the activation of microglia was closely related to the neuroinflammatory response. 34 Here, the association between microglia and the NLRP3 inflammasome in the substantia nigra could be clearly seen by immunofluorescence co-localization. As shown in Figure 7a, compared with control group, the NLRP3 inflammasome (red) was obviously activated in the areas where microglia (green) were activated in the 6-OHDA group. After H-k L. m treatment, the activation of microglia and the NLRP3 inflammasome was inhibited.
The correlation analysis between IBA-1 and NLRP3 showed that the activation of microglia was positively correlated with the expression of NLRP3 inflammasome (Figure 7b). In addition, H-k L. m was also able to suppress 6-OHDA-induced activation of the NLRP3 inflammasome signaling, including NLRP3, ASC, and caspase-1 (Figure 7c).

| H-k L. m exerted DA neuroprotection by inhibiting microglial NLRP3 inflammasome activation
To further explore whether H-k L. m-mediated DA neuroprotection was related to the inhibition of NLRP3 inflammasome activation, NLRP3 knockout mice was applied. As shown in Figure 8a  This study demonstrated that 6-OHDA could change the abundance and diversity of intestinal flora in rats, while no significant difference was exhibited in LPS-and ROT-induced PD rat models. These findings were not fully consistent with the existed studies. For example, high-throughput 16S rRNA gene sequencing revealed that gut microbial environment was apparently changed in 6-OHDA-induced mouse PD model. 36 However, the mechanisms underlying how 6-OHDA caused gut microbial environment alterations were unknown. On the other hand, previous studies showed that chronic infusion of ROT for 22-28 days induced enteric nervous system dysfunction and delayed gastric emptying, which might further affect the composition of gut microbiota. 37,38 Another evidence indicated that a bilateral intra-nigral administration of LPS caused gastric dysmotility to further influence gut microbiota, which was consistent with human PD. 39 Gastric dysmotility was closely associated with an imbalance of neurotransmitters in the dorsal motor nucleus of the vagus nerve.
Despite understanding the direct role of LPS, ROT, and 6-OHDA in PD pathophysiology, the mechanisms responsible for the early gastrointestinal tract complications associated with PD etiology was far from being elucidated. 40 Here, there are several limitations in this study. First, the time-course study on gut microbial changes would provide more detail information. Second, how 6-OHDA affected gut microbial environment in rats was unrevealed. Thus, the mechanisms underlying 6-OHDA-induced alteration of microbes warrant further exploration. This study provides a baseline for understanding the microbial communities in 6-OHDA-induced PD rat model to further investigate the role of gut microbiota in PD pathogenesis.
At the beginning, the initial design of this study was mainly focused on live L. m, in which H-k L. m was used as a negative control.
According to the current publications, the administration time of probiotics was different, including 2 weeks (probiotics treatment for 1 week previous PD and Alzheimer's disease animal model establishment and followed for further 1 week) 41 and 5 weeks (probiotics treatment for 2 weeks previous PD animal model establishment and followed for further 3 weeks). 42 Considering the premise that live probiotics should spend more time to colonize in the intestinal tract, prophylactic treatment was applied to give live L. m. However, it was unexpectedly found that H-k L. m produced neuroprotection against DA neuronal loss, whereas no significant neuroprotection was discerned after live L. m treatment. Although the underlying mechanisms remained unilluminated, it was quite interesting to investigate the reasons why H-k L. m rather than live L. m produced neuroprotection. In addition, this study was a pilot study just demonstrating pretreatment of H-k L. m could confer neuroprotection against DA neuronal loss. Next, to investigate whether post-treatment of live or H-k L. m exerted DA neuroprotection was an interesting area needs to be studied in future.
At present, the pathogenesis of PD is still unclear. However, the role of neuroinflammation in the occurrence and development of PD has been well confirmed. Microglia are brain resident immune cells, F I G U R E 6 H-k L. m attenuated 6-hydroxydopamine (6-OHDA)-induced microglial activation and release of inflammatory factors. Rat brains were collected and sectioned. Double immunofluorescence staining with anti-IBA-1 (green) and tyrosine hydroxylase (TH) (red) antibodies was performed (a). The fluorescence intensity of staining was quantified. Scale = 100 μm. Correlation analysis of TH and IBA-1 expressions was determined (b). The protein expression levels of IBA-1, IL-18, IL-1β, and TNF-α in rat midbrain were determined by western blotting (c). Data were presented as mean ± SEM from five rats. *p < 0.05 compared with control group; # p < 0.05 compared with 6-OHDA group.
F I G U R E 7 H-k L. m inhibited NLRP3 inflammasome activation in microglia. Staining with anti-NLRP3 (red) and IBA-1 (green) antibodies was performed by double immunofluorescence (a). The fluorescence intensity of staining was quantified to assess NLRP3 and IBA-1 levels. Scale = 50 μm. Correlation analysis between NLRP3 and IBA-1 expression was performed (b). The protein levels of NLRP3, ASC, pro-caspase-1, and caspase-1 in rat midbrain were determined by western blotting (c). Data were presented as mean ± SEM from five rats. *p < 0.05 compared with control group; # p < 0.05 compared with 6-hydroxydopamine (6-OHDA) group.
although they can promote neurogenesis and synaptic pruning by clearing debris to maintain brain development and the role of the steady state. In order to response from acute inflammatory cytokines and the stimulation of nerve toxicity, microglia are activated and produce various cytokines and chemokines. 43 At the same time, DA neuronal damage and loss occurred after the abnormal activation of F I G U R E 8 H-k L. m exerted dopamine (DA) neuroprotection by inhibiting microglial NLRP3 inflammasome activation. H-k L. m (1.4 Â 10 9 cfu) was administered in wild-type (WT) and NLRP3 knockout (KO) mice. Mouse behavior dysfunctions were assessed by rotarod (a) and stepping assays (b). DA neurotoxicity was assessed by tyrosine hydroxylase (TH) protein expression detection (c). Microglia activation and inflammatory mediators levels were determined by IBA-1, IL-18, IL-1β, and TNF-α protein expressions measurement (d). Data were presented as mean ± SEM from five to nine rats. *p < 0.05 compared with control group; # p < 0.05 compared with 6-hydroxydopamine (6-OHDA) group.
microglia. 44  inflammasome activation is closely related to neurodegenerative diseases, such as PD. 48 Various stimuli, such as ATP, bacterial toxins, and pathogen-associated RNA, as well as certain cellular signals, such as K + efflux, Ca 2+ mobilization, Na + influx and Cl À efflux, ROS and mitochondrial dysfunction, affect NLRP3 inflammasome activation. 49  In the present study, live L. m did not present beneficial effects.
According to the International Society for Probiotic and Probiotic Science, the premise of probiotic action is adequate administration. 54 Unfortunately, although the administration dose of this experiment is already in the category of conventional administration dose of probiotics, due to the limitations of manual operation in the laboratory, the administration dose cannot be achieved. In addition, the colonization cycle of live probiotics may also be one of the reasons why they cannot act as soon as possible compared with the inactivated bacterial microbial components directly exposed to the intestine. In this study, heat treatment resulted in the loss of biological activity of lactic acid bacteria, thus reducing the potential risk of certain viable bacteria, such as disrupted bacterial balance, disrupted risk of translocation, and bacteremia and sepsis due to translocation, especially in immunocompromised, critically ill patients and pediatric populations. 55,56 Heat-killed bacteria can exert probiotic properties under conditions that avoid the above risks. Indeed, bacterial viability or bacterial cell wall integrity is not a necessary condition for probiotics to exhibit beneficial effects. Microbial components of heat-killed bacteria, such as extracellular polysaccharides, teichoic, and lipoteichoic acids, peptidoglycan are active at signal transduction receptors in intestinal epithelium, dendritic cells, and other immune enterocytes, stimulating the innate immune system, adaptive responses, and immunomodulatory activities. 57 Therefore, although heat-killed bacteria lose their biological viability compared with live bacteria, heat-killed bacteria may still produce microbiologically similar effects in a nonmicrobiological manner. For example, heat-killed Lactobacillus fermentum and Lactobacillus delbrueckii could remodel mouse intestinal bacteria and affect the level of endocrine substance corticosterone. 58 Moreover, heat-killed Lactobacillus brevis SBC8803 enhanced mouse intestinal barrier 45 and heat-killed Lactobacillus plantarum KCTC13314BP and heat-killed Lactobacillus brevis could increase the phagocytic activity of macrophages and produce immune stimulation by activating MAPK and STAT3 or TAK1 pathways. 59,60 In addition, heat-killed lactic acid bacteria can transform T helper (Th) type 2 response to Th1 response and have immunoregulatory ability. 61,62 These beneficial effects exerted by heat-killed bacteria were not different from those generated by live probiotics. Therefore, the immunoregulatory effects of live or dead probiotics might play an important role in the communication among gut microbiota, neuroendocrine system and brain. 63,64 It has been confirmed that live probiotics could influence the gastrointestinal microbiota and have immunomodulatory effects, while dead probiotics might generate anti-inflammatory responses. 65,66 In this study, H-k L. m presented neuroprotection against DA neuronal loss, whereas no DA neuroprotection was exhibited after live L. m treatment. This phenomenon was unusually speculated in several ways: (i) in contrary to live L. m, a new paradigm was created that H-k L. m could also produce beneficial properties; (ii) H-k L. m might eliminate the risk of leakage of other live bacteria systemically to further exert neuroprotection in brain; and (iii) several kinds of active components of H-k L. m could influence the communication between gut microbiota and brain. However, the underlying mechanisms remains unclear and warrant further systematic investigation. Collectively, although the relative importance of live or dead probiotics-mediated beneficial effects is difficult to evaluate, the improved safety, longer shelf-life and relatively easy standardization are attractive advantages of dead probiotic preparations over live ones.
Until now, PD is based on drug therapy, which actually does not truly curb the degeneration of DA neurons, and long-term use of these drugs is also accompanied by serious side effects. 67